The present invention relates to a method of attaching and/or crystallizing macromolecules, to the chemical reagents used in the said method, to the products obtained as well as to the applications of the said products in the field of materials and of structural biology, in particular as biosensors or as biomaterials.
The knowledge of the structure of proteins and in particular of their active sites is essential for understanding their mechanism of action. Several methods are available for carrying out such studies: X-rays, NMR, electrocrystalography (2D crystallization).
For carrying out the crystallization proper, the technique of two-dimensional crystallization on a lipid film or monolayer, at the air/water interface (E. E. Ugziris et al., Nature, 1983, 301, 125-129), allows the formation of self-organized systems of biological macromolecules (crystals) and the determination of the structures of these molecules by electron microscopy analysis of the crystals obtained.
This method consists in creating a lipid monolayer at the level of an air/liquid interface, the lipids being selected so as to interact with the proteins, present in the liquid phase, which attach to the lipids and then form an organized network.
The attachment of the proteins to the lipids of the monolayer involves chemical interactions at the level of the polar head of the lipids. These interactions are either aspecific, the lipids possessing charged polar ends, giving rise to crystallization through ionic interactions, or specific. In the latter case, the polar head of the lipids carries ligands exhibiting high affinity with the proteins to be attached.
In particular, it has been possible to show that soluble proteins can two-dimensionally crystallize on lipid films which are charged, or which are functionalized by a ligand for the protein studied (B. J. Jap et al., Ultramicroscopy, 1992, 46, 45-84).
More recently, lipids functionalized by metal complexes such as nickel complexes (E. W. Kubalek et al., J. Struct. Biol., 1994, 113, 117-123) have made it possible to crystallize so-called histidine tagged fusion proteins. These proteins indeed possess, at their N- or C-terminal end, a sequence composed of several histidines. It has been possible to show that the attachment of such proteins to a lipid-nickel was due to a strong interaction between the nickel complex and the poly-histidine sequence (C. Vxc3xa9nien-Brian et al., J. Mol. Biol., 1997, 274, 687-692). Such functionalized lipids have made it possible to obtain crystallization, in particular in the case where an appropriate ligand was not available.
However, the crystallization of proteins on lipid films has the disadvantage of being relatively random and of depending on many factors, which are difficult to control simultaneously:
the ligand carried by the lipids should be sufficiently accessible in order to be able to interact with the proteins. This accessibility depends on the length of the spacer arm between the lipid and the ligand: too short, it gives rise to a penetration of the protein inside the lipid layer; too long, it confers an extremely high degree of freedom on the bound protein and increases the incidence of defects in the crystal;
the lipid monolayer should be sufficiently fluid in order to confer a sufficient lateral and rotational mobility on the bound protein, thus allowing the proteins to organize relative to each other and to develop intermolecular contacts, so as to give rise to the crystal;
another difficulty, inherent to crystallization on a lipid monolayer, relates to the stability of the monolayer; indeed, the stability of the air/liquid interface is difficult to control. In addition, the lipid monolayer should remain stable, not only before the attachment of the proteins, but also after their attachment, in order to allow the spatial organization of the proteins;
for the microscopy study which follows the crystallization step, it is necessary to produce a multitude of planes, because of the planar nature of the structure obtained.
Consequently, the inventors set themselves the aim of providing a method which makes it possible to attach in solution and to optionally induce self-organization of macromolecules which is more suitable for the requirements of practical use than the 2D crystallization methods previously used.
The subject of the present invention is a method for the attachment and/or self-organization of biological macromolecules, characterized in that it essentially comprises the incubation, without stirring, for at least 15 minutes, of a macromolecule in solution with nanotubes of carbon closed at their ends, under suitable temperature and pH conditions.
Nanotubes were discovered in 1991 (S. Ijima, Nature 1991, 354, 54-56); since then, they have generated a lot of interest, in particular because of their mechanical properties: high mechanical resistance (M. M. J. Treacy et al., Nature 1996, 381, pp. 678-680) and electronic properties: conductor or semiconductor property (J. W. G. Wildxc3x6er et al., Nature 1998, 391, 59-62: T. W. Odom et al., Nature, 1998, 391, 62-64).
Several methods of preparing nanotubes have been described, including that by T. W. Ebbesen et al. (Nature, 1992, 358, 220-222), which makes it possible to obtain a high yield. Methods of purifying nanotubes have also been described (H. Hiura et al., Adv. Mater., 1995, 7, 275-276; J-M Bonard et al., Adv. Mater., 1997, 9, 827-831 and G. S. Duesberg et al., Chem. Commun. 1998, 435-436); these various methods make it possible to obtain the desired quantities of nanotubes. Methods for the chemical functionalization of nanotubes of carbon have also been described (International Application PCT WO97/32571).
Other methods for the chemical functionalization of nanotubes have also been described; there may be mentioned for example TSANG S. C. et al., Journal of the Chemical Society, Chemical Communications, 1995, 17, 1803-1804 and DAVIS J. J. et al., Inorganica Chimica Acta, 1998, 272, 1, 2, 262-266.
However, they involve chemical reactions which either dramatically modify the geometry of the nanotubes (opening of the ends, partial destruction of the outer sheets), or destroy the intrinsic physical properties of the nanotubes and consequently do not allow organization of biological macromolcules such as proteins, on the nanotubes. Nanotubes modified by such destructive methods are not therefore suitable for adsorption and/or self-organization at their outer surface of synthetic products or of biological macromolecules.
Depending on the technique and the conditions used, several structures of nanotubes may be prepared: the nanotubes have in particular so-called multi-wall nanotube structures (MWNT) or single-wall nanotube structures (SWNT) of graphite. They can be completely, partially or not at all oxidized.
Thus, the nanotubes are, from a chemical point of view, polymers composed solely of carbon and which may comprise up to a million atoms. In accordance with the laws of the chemistry of carbon, the atoms of a nanotube are linked via a solid covalent bond and each atom possesses exactly three neighbours. Thus, regardless of its length, a nanotube is obliged to close at its ends, so as not to leave any chemical bond alone there. In general, its diameter is generally between 1 and 30 nm and its length may be up to several micrometers.
From a physical point of view, nanotubes can be defined as carbon crystals extending in a single direction, the repeating unit having the symmetry of a helix (B. I. Yakobson et al., American Scientist, 1997, 85, 324-337).
According to an advantageous embodiment of the said method, the said biological macromolecules are in particular soluble, membrane or transmembrane proteins, enzymes, antibodies, antibody fragments or nucleic acids.
According to another advantageous embodiment of the said method, the said nanotubes of carbon are functionalized by physical adsorption of a chemical reagent of general formula H-E-L, in which:
H represents a hydrophilic group, selected from the positively or negatively charged groups; ligands or analogues of biological macromolecules, such as, without limitation, biotin, novobiocin, retinoic acid, steroids, antigens; organometallic complexes interacting with amino acids or nucleic acids, such as complexes of copper, zinc, nickel, cobalt, chromium, platinum, palladium, iron, ruthenium or osmium with ligands such as IDA, NTA, EDTA, bipyridine or terpyridine, the said ligands being optionally functionalized with alkyl groups for bonding to E (at the level of X); positively or negatively charged groups are understood to mean, without limitation: ammoniums, carboxides, phosphates, sulphonates; the following groups may be mentioned for example: xe2x80x94N(CH3)3+ or xe2x80x94CO2xe2x80x94.
E represents a spacer arm, selected from C1-C10 carbon chains, optionally substituted with alkyl groups or otherwise, having unsaturations or polyoxyethylene units which may have or otherwise in the middle of the chain phosphate groups, such as: 
xe2x80x83in which:
m represents an integer from 1 to 10,
X represents O, NHCO, OCO, COO, CONH, S, CH2 or NH and constitutes, at the ends of the said carbon chains, organic functions for adhesion of the ester, amide, ether or thioether type;
L represents a lipid unit with one or more chains of variable length, in the form of C12-C20 having unsaturations or otherwise; an aromatic group of formula Ar1 or of formula Ar2: 
xe2x80x83in which:
A represents a hydrogen atom, one of the following groups: alkyl, CF3, NO2, NH2, OH, O-alkyl, S-alkyl, COOH, halogen, an aromatic ring or an aromatic heterocycle in the form of C4-C6, optionally polysubstituted with electron-donating groups of the alkyl type or electron-attracting groups of the CF3 or halide type; L represents for example one of the following aromatic groups: benzyl, naphthyl, anthracenyl, fluoroenyl, tetrabenzofluoroenyl, and
Y represents a bond with E.
For the purposes of the present invention, alkyl is understood to mean linear or branched or optionally substituted C1-C6 alkyl groups.
Surprisingly, both untreated (non-functionalized) nanotubes of carbon and nanotubes of carbon functionalized by non-destructive methods, as defined above, can be used in the method according to the invention.
The functionalization according to the present invention is, surprisingly, non-destructive for the nanotubes; in particular, it avoids the opening of their ends.
It is possible with such a method to adsorb and/or to self-organize at the outer surface of the nanotubes of carbon either synthetic products or biological macromolecules.
Indeed, the present invention makes it possible to induce the formation of arrangements of macromolecules (self-organization) such as proteins, with a helical symmetry.
According to another embodiment of the said method, the said solution consists of a solvent for solubilizing the said biological macromolecules, which is aqueous or aqueous-alcoholic and which optionally contains at least one detergent, depending on the biological macromolecule to be crystallized.
According to another advantageous embodiment of the said method, the incubation conditions are preferably the following: incubation at room temperature, for 15 minutes to 48 hours, at a pH of between 5.5 and 8.5.
Surprisingly, the said method makes it possible to obtain arrangements of biological macromolecules which allow structural studies by electronic microscopy and the preparation of novel nano materials which can be used for their physical, electrical or biological properties.
Such a method has the advantage of making the crystallization of proteins reproducible; in particular, it is easy, in the case where a protein does not crystallize in the presence of nanotubes of a given diameter, to use the method with nanotubes of different diameter; indeed, the crystallization of a given protein depends on the diameter of the nanotubes.
However, in the present invention, it is possible to vary the diameter of the nanotubes and to use equally well multi-wall or single-wall nanotubes of carbon which are completely, partially or not at all oxidized.
Also surprisingly, in the method according to the invention, the attachment or the crystallization of macromolecules on the nanotubes of carbon may be, under the appropriate experimental conditions, as defined above, either spontaneous, that is to say in the absence of any other synthetic products, or induced by addition of a chemical reagent H-E-L, as defined above.
Also surprisingly, the various factors which may come into play in order to allow a reproducible crystallization are, as already specified above, the following: the concentration of the samples, the choice of the solvents, the ionic restraint, the pH of the solutions, the incubation time and the diameter of the nanotubes.
Both the reagents in which L represents a lipid unit with one or more chains of variable length in the form of C12-C20, having unsaturations or otherwise, and the reagents in which L represents an aromatic group of formula Ar1 or of formula Ar2, make it possible to obtain functionalized nanotubes suitable for the arrangement of macromolecules at their surface; however, the reagents in which L represents an aromatic group of formula Ar1 or of formula Ar2 are particularly preferred.
The subject of the present invention is also bionanomaterials, characterized in that they essentially consist of nanotubes of carbon, on which biological macromolecules are attached by means of non-covalent bonds.
The subject of the present invention is also bionanomaterials, characterized in that they essentially consist of nanotubes of carbon, on which biological macromolecules are self-organized in a crystalline form.
According to an advantageous embodiment of the said bionanomaterials, they are obtained with the aid of a method as defined above.
The subject of the present invention is in addition the applications of the said bionanomaterials to the structural study of the biological macromolecules which are associated with them, as biological reagent and more particularly as immunological reagent and as biosensors or bioconductors.
The subject of the present invention is in addition a chemical reagent capable of being physically adsorbed on nanotubes of carbon, characterized in that it has the general formula H-E-L, in which:
H represents a hydrophilic group selected from the positively or negatively charged groups; ligands or analogues of biological macromolecules; organometallic complexes interacting with amino acids or nucleic acids and whose ligands are optionally functionalized with alkyl groups for bonding to E;
E represents a spacer arm, selected from C1-C10 carbon chains, optionally substituted with alkyl groups, having unsaturations or otherwise or polyoxyethylene units which may have or otherwise in the middle of the chain phosphate groups, such as: 
xe2x80x83in which:
m represents an integer from 1 to 10,
X represents O, NHCO, OCO, COO, CONH, S, CH2 or NH and constitutes, at the ends of the said carbon chains, organic functions for adhesion of the ester, amide, ether or thioether type;
L represents an aromatic group of formula Ar1 or of formula Ar2: 
xe2x80x83in which:
A represents a hydrogen atom, one of the following groups: alkyl, CF3, NO2, NH2, OH, O-alkyl, S-alkyl, COOH, halogen, an aromatic ring or an aromatic heterocycle in the form of C4-C6, the said rings being optionally polysubstituted with electron-donating groups of the alkyl type or electron-attracting groups of the CF3 or halide type; and
Y represents a bond with E.
According to an advantageous embodiment of the said chemical reagent, it has one of the following structures: 
According to another advantageous embodiment of the sa id chemical reagent, H is selected from the following organometallic complexes: 
with R1=organic group for bonding to E.
In addition to the preceding arrangements, the invention further comprises other arrangements, which will emerge from the description which follows, which refer to exemplary embodiments of the method which is the subject of the present invention as well as to the accompanying drawings, in which: